The broad and long-term goals of this proposal are to understand the morphogenetic mechanisms by which the vertebrate brain assumes its characteristic three-dimensional shape, with emphasis on formation and function of the brain ventricular system. One unique aspect of vertebrate development is that the brain forms as a tube, from which develops a system of interconnected cavities, known as the brain ventricular system. This tubular structure is conserved, likely because the cerebrospinal fluid (CSF) within the ventricles is necessary for normal brain development and function. Consistent with this suggestion, in the adult, CSF is essential for normal brain function, however, the requirement for CSF during embryogenesis is not clear. This proposal will examine the activity of genes required for brain morphogenesis and ventricle formation, using the zebrafish as a model. The zebrafish is a wonderful system with which to analyze brain morphogenesis, as forward genetic screens can define mutants defective in this process, live-imaging of the brain at single cell resolution is feasible, and sophisticated molecular assays are routine. I hypothesize that three forces are required for brain morphogenesis, and contribute to ventricle development: those intrinsic to the neuroepithelium, those extrinsic, and those resulting from active inflation of the brain lumen. In an exploratory study, we showed that brain ventricles form over a six-hour period after neural tube closure, and defined mutants defective in brain morphogenesis and ventricle formation. In this proposal, we will first, analyze the mechanisms of lumen inflation, focusing on the role of NaKATPase alpha and beta subunits, using loss of function, transgenic and mosaic assays. Second, we will analyze the mechanism by which the midline of the brain primordium opens, using mosaic, transgenic and molecular assays. We have shown that midline opening in the hindbrain requires a regulator of myosin phosphatase, Ppp1r12a. Third, we will analyze the phenotypes of three other mutants with apparent midline opening defects. These are zonderzen, atlantis and fbxo5. Fourth, we will examine the temporal requirement for embryonic CSF during primary and secondary neurogenesis, using a brain explant preparation and fluid drainage method. This study will provide new understanding of the mechanisms underlying vertebrate brain morphogenesis, and will help define the requirement for cerebrospinal fluid during neurogenesis. These data will also increase understanding of the genetic basis for the large number of brain structural defects in humans, and their associated functional abnormalities. Project Narrative: This proposal will identify genes required to build the brain into the correct three-dimensional shape, which is required for normal brain function. Abnormalities in brain structure comprise a large group of birth defects associated with mental retardation and cognitive deficits, which may include autistic disorder. [unreadable] [unreadable] [unreadable]